Elucidating epigenetic regulation of the RNA polymerase II transcription cycle

• Applicant: Dr. Jan-Hendrik Spille
• Subject Area: Biophysics ; General Genetics and Functional Genome Biology
• Term: from 2016 to 2019
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 323110857

Gene expression levels determine the protein content of a cell and thereby its phenotype. All cells in an organism carry a copy of the same genetic information. The regulation of gene expression determines the function of the cell. Misregulation can lead to a broad range of diseases. Gene expression begins with transcription. The RNA polymerase II (Pol II) holoenzyme transcribes a copy of genetic information encoded in the DNA to make it available for protein translation. Chromatin structure, chromatin modifications, and transcription factor binding all contribute to the regulation of transcription. Here, I focus on the effect of posttranslational histone modifications (PTMs). The DNA is tightly wrapped around histone octamers. Modifying amino acid residues by e.g. acetylation or methylation changes the DNA-histone interaction and can create binding surfaces for transcription factors. PTMs are heritable traits and thus form an epigenetic memory that is not encoded in the DNA itself. Specific modifications are associated with the transcriptional state of a gene. Preliminary data shows that Pol II forms clusters of dozens of molecules at actively transcribed genes with a lifetime of a few seconds. Cluster lifetime determines mRNA output at a specific gene locus. Our data also show that inhibition of histone deacetylation leads to an increase in cluster lifetime by an order of magnitude. In this research project, I will study the effect of PTMs on the kinetics of Pol II clustering and mRNA synthesis. Standard assays for probing protein-DNA interaction and mRNA levels work with large populations of chemically fixed cells and therefore fail to detect dynamics and transient interactions. Live cell fluorescence microscopy can fill in this gap. I will use lattice light sheet microscopy (LLS) to investigate the kinetics of the Pol II transcription cycle with high temporal resolution. Live cell compatible antibody fragments and rapid multicolor imaging will indicate the PTM state at sites of Pol II clustering. The superior imaging capabilities of LLS will allow for a detailed understanding of cluster properties like assembly and disassembly kinetics, lifetime, size, and the fraction of polymerases recruited to the DNA. Simultaneously detected mRNA synthesis at a specific, single copy gene locus in live cells will reveal variability in the deterministic relation between cluster lifetime and mRNA production with important implications for understanding the mechanism of transcriptional bursting. I will develop a highly multiplexed live cell DNA FISH assay to investigate promoter-dependent differences in this behavior and to study differential effects of globally induced PTMs on Pol II dynamics at up- and downregulated gene loci in a single nucleus. Finally, I will introduce histone modifications at specific sites in the promoter region of a silenced gene and follow the kinetics of its epigenetically induced activation.

• DFG Programme: Research Fellowships
• International Connection: USA
• Host: Professor Ibrahim Cisse, Ph.D.